NLRP3; NF-κB
RAGE (receptor for advanced glycation end products); MAPK pathway components including p38, JNK, and ERK1/2; NF-κB p65; NLRP3 inflammasome [1]

Advanced glycation end products (AGEs) could interact with the receptor for AGE (RAGE) as a sterile danger signal to induce inflammation. 4′-methoxyresveratrol (4′MR), a polyphenol derived from Dipterocarpaceae, has not been studied for its anti-inflammation effects. In the present study, we sought to explore the protective role of 4′MR in AGEs-induced inflammatory model using RAW264.7 macrophages. 4′MR significantly inhibited gene expression of pro-inflammatory cytokines and chemokines, such as interleukin 1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α) and monocyte chemoattractant protein-1 (MCP-1), as well as two typical pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). Besides, 4′MR significantly decreased oxidative stress, demonstrated by levels of ROS production, protein carbonyl and advanced oxidation protein product via down-regulation of NADPH oxidase. Further analysis showed that 4′MR attenuated the RAGE overexpression induced by MGO-BSA. It also blocked the downstream signal of AGE-RAGE, particularly, MAPKs including p38 and JNK, and subsequently reduced NF-κB activation. Additionally, 4′MR significantly abated the activation of NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome including NLRP3 and cleaved caspase-1 and reduced the secretion of mature IL-1β. Taken together, our results suggest that the anti-inflammatory effect of 4′MR is mainly through suppressing RAGE-mediated MAPK/NF-κB signaling pathway and NLRP3 inflammasome activation. 4′MR could be a novel therapeutic agent for inflammation-related diseases.[1]

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At 10 μM, 4'-Methoxyresveratrol significantly inhibited mRNA expression of pro-inflammatory cytokines and chemokines (IL-1β, IL-6, TNF-α, MCP-1) as well as pro-inflammatory enzymes (iNOS and COX-2) induced by AGEs (200 μg/mL MGO-BSA) in RAW264.7 macrophages after 24 h treatment [1]
- The compound reduced nitric oxide (NO) level by approximately 2.04-fold in the supernatant of AGEs-treated cells (p < 0.05) at 24 h [1]
- 4'-Methoxyresveratrol (10 μM) decreased reactive oxygen species (ROS) production by about 43% (p < 0.05), advanced oxidation protein product (AOPP) by 32% (p < 0.001), and protein carbonyls by 15% (p < 0.05) compared to AGEs-treated group at 24 h [1]
- The compound down-regulated mRNA expression of NADPH oxidase subunits NOX1 and NOX2 by approximately 35.4% (p < 0.05) and 25.8% (p < 0.05), respectively, at 24 h [1]
- 4'-Methoxyresveratrol (10 μM) attenuated AGE-induced overexpression of RAGE at both mRNA and protein levels at 24 h (p < 0.05) [1]
- At 45 min treatment, the compound significantly inhibited AGE-induced phosphorylation of p38 MAPK (p < 0.05), JNK (p < 0.05), and NF-κB p65 (p < 0.05), but did not affect ERK1/2 phosphorylation [1]
- 4'-Methoxyresveratrol (10 μM, 24 h) reduced AGE-induced protein levels of NLRP3 by approximately 1.85-fold and cleaved caspase-1 by approximately 2.04-fold (p < 0.05) [1]
- The compound attenuated AGE-induced secretion of mature IL-1β in culture supernatant at 24 h as measured by ELISA [1]

Intracellular ROS Production Measurement[1]
The effect of 4'-O-Methylresveratrol on ROS production was determined by a method according to our previous study. For quantification of intracellular ROS level, RAW264.7 macrophages were plated in a 96-well plate for 24 h, and then further stimulated with 200 µg/mL BSA or MB with or without 10 µM of 4'-O-Methylresveratrol for 24 h. After the cultivation, supernatant was aspirated and cells were washed by warm PBS twice. Then cells were incubated with 25 µM 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) in DF10 for 1 h at 37 °C. After that, cells were washed with warm PBS twice. Finally, the fluorescence intensity of DCFH-DA-activated cells was determined by emission at 485 nm and excitation at 528 nm employing a microplate reader after adding 200 µL HBSS to each well. Results were expressed as a percentage of non-glycated BSA.

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Protein Carbonyl and AOPP Levels Measurement[1]
After treatment with 200 µg/mL BSA or MB with or without 10 µM 4'-O-Methylresveratrol for 24 h, lysates from the cell culture were prepared using a homogenizer. The concentration of total protein was measured by a BCA protein assay kit. Protein carbonyl expressed as nmol per mg total protein measured by a Protein Carbonyl Assay Kit according to the manufacturer’s protocol. The content of advanced oxidation protein products (AOPP) was determined as described by our previous study. In brief, the standard curve was draw by 0, 10, 20, 40, 80, and 100 µmol/L chloramine-T and 300 µL of protein lysates were used for sample detection. Seventy-five microliters of 1.16 M KI and 150 µL of acetic acid were added into the tubes which served as reactants with chloramine-T or samples. After that, their absorbance was measured by microplate reader at 340 nm immediately. The AOPP quantity was calculated with respect to chloramine-T and results showed in nmol of chloramine-T equivalent per mg total protein.

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RNA Isolation and qPCR Analysis[1]
RAW264.7 macrophages (106 cells/well) were cultured in DF10 supplemented with 200 µg/mL BSA or MB with or without 10 µM 4'-O-Methylresveratrol for 24 h, the total RNAs were extracted with a standard TRIzol method according to the manufacturer’s instructions. The concentrations of extracted RNAs were measured by a spectrophotometer. The cDNA synthesis was performed with reverse transcription by a PrimeScript RT reagent Kit according to the manufacturer’s instructions. A real-time PCR quantitation of target genes was performed using QuantiFast SYBR-Green RT-PCR kits. 18S expression was used as an internal control. The sequences of forward and revere primers used are shown in Appendix A (Table A1). The measurements were analyzed using the ΔCT method with LightCycler 96 in the qPCR apparatus.

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Measurement of NO and IL-1β Levels[1]
RAW264.7 macrophages (106 cells/well) were cultured in DF10 supplemented with 200 µg/mL BSA or MB with or without 10 µM 4'-O-Methylresveratrol for 24 h, and supernatants were collected for nitric oxide (NO) and interleukin 1β (IL-1β) measurement. Levels of NO and IL-1β were determined by a mouse NO and IL-1β ELISA kit according to the manufacturer’s instructions.

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Protein Extraction and Western Blot Analysis[1]
RAW264.7 macrophages (106 cells/well) were cultured in DF10 supplemented with 200 µg/mL BSA or MB with or without 10 µM 4'-O-Methylresveratrol for 45 min after being starved for 4 h in six-well dishes. Then, cells were washed with pre-cooled PBS twice and lysed with 200 µL of RIPA lysis buffer on ice for 15 min. The lysates were subsequently transferred into 2 mL tubes for collecting supernatant by centrifugation (12,000× g, 10 min, 4 °C). The concentration of protein was determined by a BCA protein assay kit. A total of 30 µg protein was run per lane on SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked for 1 h at room temperature by 5% fat-free milk and incubated with primary antibodies (ERK1/2, p-ERK1/2, JNK, p-JNK, p38 MAPK, p-p38 MAPK, p65, p-p65, NLRP3, and cleaved caspase-1 were used at 1:1000; RAGE and β-actin were used at 1:3000) overnight at 4 °C accompanied by soft shaking. After washing 3 times per 5 min with PBST, the PVDF membranes were then incubation for 30 min by a HRP-conjugated secondary antibody (1:1000) at room temperature. Finally, immunoreactive bands were visualized with enhanced chemiluminescence (ECL) reagents and detected by ChemiDoc® MP Image Lab. The image Lab Software was used for quantifying band densities.

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Cell culture: Mouse RAW264.7 macrophages were maintained in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in 5% CO2 atmosphere. No significant cytotoxic effect of 4'-Methoxyresveratrol was observed below 30 μM, and 10 μM was used in all experiments [1]
- Preparation of AGEs: Methylglyoxal-modified bovine serum albumin (MGO-BSA) was prepared by incubating 10 mg/mL endotoxin-free BSA with 55 mM methylglyoxal at 37°C for six days, followed by dialysis against PBS for two days at 4°C and filtration through 0.22 μm filter membrane. The fluorescence intensity of MGO-BSA was 70-fold higher than that of BSA (excitation 370 nm, emission 440 nm) [1]
- ROS measurement: Cells were treated with 200 μg/mL BSA or MGO-BSA with or without 10 μM 4'-Methoxyresveratrol for 24 h, then incubated with 25 μM DCFH-DA in medium for 1 h at 37°C. Fluorescence intensity was measured at excitation 485 nm and emission 528 nm using a microplate reader. Results were expressed as percentage of non-glycated BSA control [1]
- Protein carbonyl and AOPP measurement: Cell lysates were prepared after 24 h treatment. Total protein concentration was determined by BCA assay. Protein carbonyl levels were measured using a commercial kit. AOPP levels were determined by a chemical method: 300 μL protein lysates were mixed with 75 μL 1.16 M KI and 150 μL acetic acid, and absorbance was measured at 340 nm immediately. Chloramine-T (0-100 μmol/L) was used as standard. Results were expressed as nmol chloramine-T equivalent per mg total protein [1]
- RNA isolation and qPCR: Total RNA was extracted using TRIzol method after 24 h treatment. cDNA was synthesized using reverse transcription kit. Real-time PCR was performed using SYBR-Green kits with 18S as internal control. Primer sequences for TNF-α, IL-6, IL-1β, MCP-1, COX-2, iNOS, NOX1, NOX2, RAGE, and 18S were provided. The ΔCT method was used for analysis [1]
- NO and IL-1β measurement: After 24 h treatment, culture supernatants were collected. NO and IL-1β levels were determined by mouse-specific ELISA kits according to manufacturer's instructions [1]
- Protein extraction and Western blot: Cells were starved for 4 h then treated for 45 min (for MAPK/NF-κB) or 24 h (for RAGE, NLRP3, cleaved caspase-1). Cells were lysed with RIPA buffer on ice for 15 min, centrifuged at 12,000×g for 10 min at 4°C. Protein concentration was determined by BCA assay. 30 μg protein per lane was separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk, incubated with primary antibodies (ERK1/2, p-ERK1/2, JNK, p-JNK, p38 MAPK, p-p38 MAPK, p65, p-p65, NLRP3, cleaved caspase-1 at 1:1000; RAGE and β-actin at 1:3000) overnight at 4°C, then with HRP-conjugated secondary antibody (1:1000) for 30 min at room temperature. Bands were visualized by enhanced chemiluminescence and quantified using imaging software [1]

[1]. 4'-Methoxyresveratrol Alleviated AGE-Induced Inflammation via RAGE-Mediated NF-κB and NLRP3 Inflammasome Pathway. Molecules. 2018 Jun 14;23(6).

5-[2-(4-methoxyphenyl)vinyl]benzene-1,3-diol is a stilbene compound. (E)-5-(4-methoxystyryl)benzene-1,3-diol has been reported in ginger, dragon's blood tree, and other organisms for which data are available.

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4'-Methoxyresveratrol (3,5-dihydroxy-4'-methoxystilbene) is a plant stilbene derived from Dipterocarpaceae and Gnetaceae families. It has been previously reported to show antiandrogenic activity in prostate cancer cells and antifungal activity in vitro, but no previous report on its anti-inflammatory effects existed before this study [1]
- The anti-inflammatory mechanism of 4'-Methoxyresveratrol is mainly through suppressing RAGE-mediated MAPK/NF-κB signaling pathway (involving p38 and JNK but not ERK) and NLRP3 inflammasome activation, thereby reducing oxidative stress and pro-inflammatory gene expression. The compound also reduces ROS production partly via down-regulation of RAGE and subsequent NOX expression [1]
- The compound may have greater bioavailability and antioxidant capacity compared to resveratrol due to the methoxyl group on the stilbene structure [1]

C15H14O3

242.26986

242.094

C, 74.36; H, 5.82; O, 19.81

33626-08-3

33626-08-3

6255462

White to off-white solid powder

1.252

446.5±14.0 °C at 760 mmHg

223.8±20.1 °C

0.0±1.1 mmHg at 25°C

1.692

3.62

2

3

3

18

259

0

COC1=CC=C(C=C1)C=CC1=CC(O)=CC(O)=C1

IHVRWFJGOIWMGC-NSCUHMNNSA-N

InChI=1S/C15H14O3/c1-18-15-6-4-11(5-7-15)2-3-12-8-13(16)10-14(17)9-12/h2-10,16-17H,1H3/b3-2+

5-[(E)-2-(4-methoxyphenyl)ethenyl]benzene-1,3-diol

4'-O-Methylresveratrol; 33626-08-3; (E)-5-(4-Methoxystyryl)benzene-1,3-diol; Desoxyrhapontigenin; RESVERATROL 4'-METHYL ETHER; 4'O-Methylresveratrol; 4-Methoxyresveratrol; Deoxyrhapontigenin; 3,5-Dihydroxy-4'-methoxystilbene;

2934.99.03.00

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 48~50 mg/mL (198.1~206.4 mM)
Ethanol: ~48 mg/mL (~198.1 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (10.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)